Andrew A. George

β-Amyloid Directly Inhibits Human α4β2-Nicotinic Acetylcholine Receptors Heterologously Expressed in Human SH-EP1 Cells

Amyloid-β (Aβ) accumulation and aggregation are thought to contribute to the pathogenesis of Alzheimers disease (AD). In AD, there is a selective decrease in the numbers of radioligand binding sites corresponding to the most abundant nicotinic acetylcholine receptor (nAChR) subtype, which contains human α4 and β2 subunits (hα4β2-nAChR). However, the relationships between these phenomena are uncertain, and effects of Aβ on hα4β2-nAChR function have not been investigated in detail. We first confirmed expression of hα4 and hβ2 subunits as messenger RNA in transfected, human SHEP1 cells by reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses. Immunoprecipitation Western analyses confirmed α4 and β2 subunit protein expression and co-assembly. Whole cell current recording demonstrated heterologous expression in SH-EP1-hα4β2 cells of functional hα4β2-nAChRs with characteristic responses to nicotinic agonists or antagonists. Nicotine-induced whole cell currents were suppressed by Aβ1-42 in a dose-dependent manner. Functional inhibition was selective for Aβ1-42 compared with the functionally inactive, control peptide Aβ40-1.Aβ1-42-mediated inhibition of hα4β2-nAChR function was non-competitive, voltage-independent, and use-independent. Pre-loading of cells with guanyl-5′-yl thiophosphate failed to prevent Aβ1-42-induced inhibition, suggesting that down-regulation of hα4β2-nAChR function by Aβ1-42 is not mediated by nAChR internalization. Sensitivity to Aβ1-42 antagonism at 1 nm was evident for hα4β2-nAChRs, but not for heterologously expressed human α7-nAChRs, although both nAChR subtypes were functionally inhibited by 100 nm Aβ1-42, with the magnitude of functional block being higher for 100 nm Aβ1-42 acting on hα7-nAChRs. These findings suggest that hα4β2-nAChRs are sensitive and perhaps pathophysiologically relevant targets for Aβ neurotoxicity in AD.

α7β2 Nicotinic Acetylcholine Receptors Assemble, Function,and Are Activated Primarily via Their α7-α7 Interfaces

We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of α7 and β2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged α7 and β2 subunits colocalize. Förster resonance energy transfer between fluorescently tagged subunits strongly suggested that α7 and β2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function α7 and β2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, α7β2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of α7 nAChRs, although amplitudes of α7β2 nAChR-mediated, agonist-evoked currents were generally ∼2-fold lower than those for α7 nAChRs. It is noteworthy that α7β2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-β-erythroidine that was not observed for α7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the α7-β2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower α7β2 nAChR current amplitudes and challenges in identifying the function of native α7β2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of β2 subunits within the α7β2 nAChRs.

The Novel α7β2-Nicotinic Acetylcholine Receptor Subtype Is Expressed in Mouse and Human Basal Forebrain: Biochemical and Pharmacological Characterization

We examined α7β2-nicotinic acetylcholine receptor (α7β2-nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric α7-nAChRs and α7β2-nAChRs. α-Bungarotoxin affinity purification or immunoprecipitation with anti-α7 subunit antibodies (Abs) was used to isolate nAChRs containing α7 subunits from mouse or human brain samples. α7β2-nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using β2 subunit–specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (α7)5-nAChRs or for homomeric α7-nAChRs constituted from unlinked α7 subunits. Pharmacological profiles were similar for (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at α7β2- versus α7-nAChRs. This study represents the first direct confirmation of α7β2-nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of α7β2-nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that α7β2-nAChR subunit isoforms with different α7/β2 subunit ratios have similar pharmacological profiles to each other and to α7 homopentameric nAChRs. This supports the hypothesis that α7β2-nAChR agonist activation predominantly or entirely reflects binding to α7/α7 subunit interface sites.

Function of Human α3β4α5 Nicotinic Acetylcholine Receptors Is Reduced by the α5(D398N) Variant

Background: The naturally occurring α5(D398N) variant alters smoking behavior, but functional differences have not been detected between α3β4α5 nAChR harboring these variants. Results: ACh-induced α3β4α5 nAChR function is lower when α5(Asn-398) substitutes for α5(Asp-398). Conclusion: The α5 variant-induced change in α3β4α5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism. Significance: α3β4α5 nAChR function may be a useful target for smoking cessation pharmacotherapies. Genome-wide studies have strongly associated a non-synonymous polymorphism (rs16969968) that changes the 398th amino acid in the nAChR α5 subunit from aspartic acid to asparagine (D398N), with greater risk for increased nicotine consumption. We have used a pentameric concatemer approach to express defined and consistent populations of α3β4α5 nAChR in Xenopus oocytes. α5(Asn-398; risk) variant incorporation reduces ACh-evoked function compared with inclusion of the common α5(Asp-398) variant without altering agonist or antagonist potencies. Unlinked α3, β4, and α5 subunits assemble to form a uniform nAChR population with pharmacological properties matching those of concatemeric α3β4* nAChRs. α5 subunit incorporation reduces α3β4* nAChR function after coinjection with unlinked α3 and β4 subunits but increases that of α3β4α5 versus α3β4-only concatemers. α5 subunit incorporation into α3β4* nAChR also alters the relative efficacies of competitive agonists and changes the potency of the non-competitive antagonist mecamylamine. Additional observations indicated that in the absence of α5 subunits, free α3 and β4 subunits form at least two further subtypes. The pharmacological profiles of these free subunit α3β4-only subtypes are dissimilar both to each other and to those of α3β4α5 nAChR. The α5 variant-induced change in α3β4α5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism.

The prototoxin LYPD6B modulates heteromeric α3β4-containing nicotinic acetylcholine receptors, but not α7 homomers

Prototoxins are a diverse family of membrane‐tethered molecules expressed in the nervous system that modulate nicotinic cholinergic signaling, but their functions and specificity have yet to be completely explored. We tested the selectivity and efficacy of leukocyte antigen, PLAUR (plasminogen activator, urokinase receptor) domain‐containing (LYPD)‐6B on α3β4‐, α3α5β4‐, and α7‐containing nicotinic acetylcholine receptors (nAChRs). To constrain stoichiometry, fusion proteins encoding concatemers of human α3, β4, and α5 (D and N variants) subunits were expressed in Xenopus laevis oocytes and tested with or without LYPD6B. We used the 2‐electrode voltage‐clamp method to quantify responses to acetylcholine (ACh): agonist sensitivity (EC50), maximal agonist‐induced current (Imax), and time constant (τ) of desensitization. For β4‐α3‐α3‐β4‐α3 and β4‐α3‐β4‐α3‐α3, LYPD6B decreased EC50 from 631 to 79 μM, reduced Imax by at least 59%, and decreased τ. For β4‐α3‐α5D‐β4‐α3 and β4‐α3‐β4‐α‐α5D, LYPD6B decreased Imax by 63 and 32%, respectively. Thus, LYPD6B acted only on (α3)3(β4)2 and (α3)2(α5D)(β4)2 and did not affect the properties of (α3)2(β4)3, α7, or (α3)2(α5N) (β4)2 nAChRs. Therefore, LYPD6B acts as a mixed modulator that enhances the sensitivity of (α3)3(β4)2 nAChRs to ACh while reducing ACh‐induced whole‐cell currents. LYPD6B also negatively modulates α3β4 nAChRs that include the α5D common human variant, but not the N variant associated with nicotine dependence.—Ochoa, V., George, A. A., Nishi, R., Whiteaker, P., The prototoxin LYPD6B modulates heteromeric α3β4‐containing nicotinic acetylcholine receptors, but not α7 homomers. FASEB J. 30, 1109–1119 (2016). www.fasebj.org